Monoclonal antibodies or variants thereof will represent a high proportion of new medicines launched in the 21st century. Monoclonal antibody therapy is already accepted as a preferred route for the treatment for rheumatoid arthritis and Crohn's disease and there is impressive progress in the treatment of cancer. Antibody-based products are also in development for the treatment of cardiovascular and infectious diseases. Most marketed monoclonal antibody products recognize and bind a single, well-defined epitope on the target ligand (eg TNFa). Manufacture of human monoclonal antibodies for therapy remains dependent on mammalian cell culture. The assembly of a complex consisting of two heavy chains and two light chains (the H2L2 complex) and subsequent post-translational glycosylation processes preclude the use of bacterial systems. Production costs and capital costs for antibody manufacture by mammalian cell culture are high and threaten to limit the potential of antibody based therapies in the absence of acceptable alternatives. A variety of transgenic organisms are capable of expressing fully functional antibodies. These include plants, insects, chickens, goats and cattle but none as yet has been used to manufacture marketed therapeutic products.
Functional antibody fragments can be manufactured in E. coli but the product generally has low serum stability unless pegylated during the manufacturing process.
Bispecific antibody complexes are engineered Ig-based molecules capable of binding two different epitopes on the either the same or different antigens. Bispecific binding proteins incorporating antibodies alone or in combination with other binding agents show promise for treatment modalities where captured human immune functions elicit a therapeutic effect, for example the elimination of pathogens (Van Spriel et al., (1999) J. Infect. Diseases, 179, 661-669; Tacken et al., (2004) J. Immunol., 172, 4934-4940; U.S. Pat. No. 5,487,890), the treatment of cancer (Glennie and van der Winkel, (2003) Drug Discovery Today, 8, 503-5100); and immunotherapy (Van Spriel et al., (2000) Immunol. Today, 21, 391-397; Segal et al., (2001) J Immunol. Methods, 248, 1-6; Lyden et al., (2001) Nat. Med., 7, 1194-1201).
Manufacturing issues are compounded where a bi-specific antibody product is based on two or more H2L2 complexes. For example, co-expression of two or more sets of heavy and light chain genes can result in the formation of up to 10 different combinations, only one of which is the desired heterodimer (Suresh et al., (1986) Methods Enzymol., 121, 210-228).
To address this issue, a number of strategies have been developed for the production in mammalian cells of full length bispecific IgG formats (BsIgG) which retain heavy chain effector function. BsIgGs require engineered “knob and hole” heavy chains to prevent heterodimer formation and utilise identical L-chains to avoid L-chain mispairing (Carter, (2001) J. Immunol. Methods, 248, 7-15). Alternative chemical cross-linking strategies have also been described for the production of complexes from antibody fragments each recognizing different antigens (Ferguson et al., (1995) Arthritis and Rheumatism, 38, 190-200) or the cross-linking of other binding proteins, for example collections, to antibody fragments (Tacken et al., (2004) J. Immunol., 172, 4934-4940).
The development of diabodies or mini antibodies (BsAb) generally lacking heavy chain effector functions also overcomes heterodimer redundancy. These comprise minimal single chain antibodies incorporating VH and VL binding sites (scFv) which subsequently fold and dimerise to form a divalent bispecific antibody monovalent to each of their target antigens (Holliger et al., (1993) PNAS, 90, 6444-6448; Muller et al., (1998) FEBS Lett., 422, 259-264). In one instance, CH1 and L-constant domains have been used as heterodimerisation domains for bi-specific mini-antibody formation (Muller et al., (1998) FEBS Lett., 259-264). A variety of recombinant methods based on E. coli expression systems have been developed for the production of BsAbs (Hudson, (1999) Curr. Opin. Immunol., 11, 548-557), though it would appear that the cost and scale of production of clinical grade multivalent antibody material remains the primary impediment to clinical development (Segal et al., (2001) J. Immunol. Methods, 248, 1-6).
Recently, the BsAb concept has been extended to encompass Di-diabodies, tetravalent bispecific antibodies where the VH and VL domains on each H and L chain have been replaced by engineered pairs of scFv binding domains. Such constructs, whilst complex to engineer, can be assembled in mammalian cells in culture in the absence of hetero-dimer redundancy (Lu et al., (2003) J. Immunol. Methods, 279, 219-232).
The structure of immunoglobulins is well known in the art. Most natural immunoglobulins comprise two heavy chains and two light chains. The heavy chains are joined to each other via disulphide bonds between hinge domains located approximately half way along each heavy chain. A light chain is associated with each heavy chain on the N-terminal side of the hinge domain. Each light chain is normally bound to its respective heavy chain by a disulphide bond close to the hinge domain.
When an Ig molecule is correctly folded, each chain folds into a number of distinct globular domains joined by a more linear polypeptide sequence. For example, the light chain folds into a variable (VL) and a constant (CL) domain. Heavy chains have a single variable domain VH, adjacent the variable domain of the light chain, a first constant domain, a hinge domain and two or three further constant domains. Interaction of the heavy (VH) and light (VL) chain variable domains results in the formation of an antigen binding region (Fv). Generally, both VH and VL are required for antigen binding, although heavy chain dimers and amino-terminal fragments have been shown to retain activity in the absence of light chain (Jaton et al., (1968) Biochemistry, 7, 4185-4195).
With the advent of new molecular biology techniques, the presence of heavy chain-only antibody (devoid of light chain) was identified in B-cell proliferative disorders in man (Heavy Chain Disease) and in murine model systems. Analysis of heavy chain disease at the molecular level showed that mutations and deletions at the level of the genome could result in inappropriate expression of the heavy chain CH1 domain, giving rise to the expression of heavy chain-only antibody lacking the ability to bind light chain (see Hendershot et al., (1987) J. Cell Biol., 104, 761-767; Brandt et al., (1984) Mol. Cell. Biol., 4, 1270-1277).
Separate studies on isolated human VH domains derived from phage libraries demonstrated antigen-specific binding of VH domains but these VH domains proved to be of low solubility. Furthermore, it was suggested that the selection of human VH domains with specific binding characteristics displayed on phage arrays could form the building blocks for engineered antibodies (Ward et al., (1989) Nature, 341, 544-546).
Studies using other vertebrate species have shown that camelids, as a result of natural gene mutations, produce functional IgG2 and IgG3 heavy chain-only dimers which are unable to bind light chain due to the absence of the CH1 light chain-binding region (Hamers-Casterman et al., (1993) Nature, 363, 446-448) and that species such as shark produce a heavy chain-only-like binding protein family, probably related to the mammalian T-cell receptor or immunoglobulin light chain (Stanfield et al., (2004) Science, 305, 1770-1773).
A characterising feature of the camelid heavy chain-only antibody is the camelid VH domain, which provides improved solubility relative to the human VH domain. Human VH may be engineered for improved solubility characteristics (see Davies and Riechmann, (1996) Protein Eng., 9 (6), 531-537; Lutz and Muyldermans, (1999) J. Immuno. Methods, 231, 25-38) or solubility maybe be acquired by natural selection in vivo (see Tanha et al., (2001) J. Biol. Chem., 276, 24774-24780). However, where VH binding domains have been derived from phage libraries, intrinsic affinities for antigen remain in the low micromolar to high nanomolar range, in spite of the application of affinity improvement strategies involving, for example, affinity hot spot randomisation (Yau et al., (2005) J. Immunol. Methods, 297, 213-224).
Camelid VH antibodies are also characterised by a modified CDR3 loop. This CDR3 loop is, on average, longer than those found in non-camelid antibodies and is a feature considered to be a major influence on overall antigen affinity and specificity, which compensates for the absence of a VL domain in the camelid heavy chain-only antibody species (Desmyter et al., (1996) Nat. Struct. Biol., 3, 803-811, Riechmann and Muyldermans, (1999) J. Immunol. Methods, 23, 25-28).
Recent structural studies on camelid antibody suggests that antibody diversity is largely driven by in vivo maturation processes with dependency on V(D)J recombination events and somatic mutation, (De Genst et al., (2005) J. Biol. Chem., 280 (14), 14114-14121).
Recently, methods for the production of heavy-chain-only antibodies in transgenic mammals have been developed (see WO02/085945 and WO02/085944). Functional heavy chain-only antibody of potentially any class (IgM, IgG, IgD, IgA or IgE) and derived from any mammal (including man) can be produced from transgenic mammals (preferably mice) as a result of antigen challenge.
The normal immunoglobulin heavy chain locus comprises a plurality of V gene segments, a number of D gene segments and a number of J gene segments. Each V gene segment encodes from the N terminal almost to the C terminal of a V domain. The C terminal end of each V domain is encoded by a D gene segment and a J gene segment. VDJ rearrangement in B-cells followed by affinity maturation provides VH binding domains which then, with VL binding domains, form an antigen recognition or binding site. Interaction of the heavy and light chains is facilitated by the CH1 region of the heavy chain and the x or X region of the light chain.
For the production of heavy chain-only antibody, the heavy chain locus in the germline comprises gene segments encoding some or all of the possible constant regions. During maturation, a re-arranged VH binding domain is spliced onto the CH2 constant region-encoding segment, to provide a re-arranged gene encoding a heavy chain which lacks a CH1 domain and is therefore unable to associate with an immunoglobulin light chain.
Heavy chain-only monoclonal antibodies can be recovered from B-cells of the spleen by standard cloning technology or recovered from B-cell mRNA by phage display technology (Ward et al., (1989) Nature, 341, 544-546). Heavy chain-only antibodies derived from camelids or transgenic animals are of high affinity. Sequence analysis of normal H2L2 tetramers demonstrates that diversity results primarily from a combination of VDJ rearrangement and somatic hypermutation (Xu and Davies, (2000) Immunity, 13, 37-45). Sequence analysis of expressed heavy chain-only mRNA, whether produced in camelids or transgenic animals, supports this observation (De Genst et al., (2005) J. Biol. Chem., 280, 14114-14121).
An important and common feature of natural camelid and human VH regions is that each region binds as a monomer with no dependency on dimerisation with a VL region for optimal solubility and binding affinity. These features have previously been recognised as particularly suited to the production of blocking agents and tissue penetration agents.
Homo- or hetero-dimers can also be generated by enzymatic cleavage of heavy chain-only antibodies or by synthetic routes (Jaton et al., (1968) Biochemistry, 7, 4185-4195 and US2003/0058074 A1). However the benefits of a monomeric antibody binding domain have yet to be used to advantage in design of multimeric proteins as reagents, therapeutics and diagnostics.
Human VH or camelid VHH produced by phage display technology lacks the advantage of improved characteristics as a result of somatic mutations and the additional diversity provided by D and J region recombination in the CDR3 region of the normal antibody binding site (Xu and Davies, (2000) Immunity, 13, 37-45). Camelid VHH, whilst showing benefits in solubility relative to human VH, is antigenic in man and must be generated by immunisation of camelids or by phage display technology.
The incorporation of VH binding domains has clear advantage over the use of scFvs which must be engineered from VH and VL domains with the associated potential of loss of specificity and avidity. VH binding domains derived from related gene families such as T-cell receptors or the shark immunoglobulin family also provide alternatives to scFv for the generation of bi- or multi-specific binding molecules. Other naturally occurring binding proteins and domains thereof including, for example, soluble receptor fragments may also be used.
Antibody classes differ in their physiological function. For example, IgG plays a dominant role in a mature immune response. IgM is involved in complement fixing and agglutination. IgA is the major class of Ig in secretions—tears, saliva, colostrum, mucus—and thus plays a role in local immunity. The inclusion of class-specific heavy chain constant regions when engineering multivalent binding complexes provides the therapeutic benefits of effector function in vivo dependent on the functionality required. Engineering of individual effector regions can also result in the addition or deletion of functionality (Van Dijk and van der Winkel, Curr. Opin. Chem. Biol., (2001) August 5 (4), 368-374). It seems likely that the optimal production and selection of heavy chain-only antibodies comprising high affinity VH binding domains (whether of human or camelid or other origin) will benefit from alternative approaches to those dependent on selection from randomised phage libraries which do not facilitate in vivo recombination and affinity maturation.
Thus, the inclusion of IgA constant region functionality would provide improved mucosal function against pathogens (Leher et al., (1999) Exp. Eye. Res., 69, 75-84), whilst the presence of IgG1 constant region functionality provides enhanced serum stability in vivo. The presence of heavy chain CH2 and CH3 constant domains provides the basis for stable dimerisation as seen in natural antibodies, and provides recognition sites for post-translational glycosylation. The presence of CH2 and CH3 also allows for secondary antibody recognition when bispecific and multivalent complexes are used as reagents and diagnostics.
Isolated, pre-rearranged camelid heavy chain-only variable region sequences have previously been cloned in front of a hinge region and human IgG1 effector domain, inserted into vectors and expressed in COS cells to generate antibody. The antibodies expressed in this in vitro environment have already undergone the processes of class (isotype) switching and affinity maturation (hypermutation) in vivo in the camel and can bind to antigen (Riechmann and Muyldermans, (1999) J. Immunol. Methods, 231, 25-38).
There remains a need in the art to maximise heavy chain-only antibody diversity and B-cell response in vivo and, in particular, to generate a functional repertoire of class specific human heavy chain-only antibodies and functional VH heavy chain-only binding domains which retain maximum antigen-binding potential for use in diverse clinical, industrial and research applications.
There also remains a need in the art to produce a soluble, bi-valent or multi-valent polypeptide binding complex comprising at least part of an antibody heavy chain, alone or in combination with an effector (light) chain, which is physiologically stable and has effector function.